The protein interactions of the BCL-2 family represent critical control points of the cell death pathway, which dictates cellular fate. Deregulation of BCL-2 family interactions is a hallmark of cancer and resistance to chemotherapy, and thus our ability to decode and restore the death pathway will have important therapeutic implications. The current funding period extended the cell death pathway by using protein interaction to define which cell deaths operate through BH3- only sentinel proteins, identify BAX/BAK as an essential gateway to mitochondrial apoptosis, and demonstrate the impact of BAX/BAK activation on mitochondrial morphology, reorganization, and dysfunction. The biological mechanisms by which the executioner proteins BAX/BAK are regulated remain hotly debated and many critical questions regarding their function are unanswered. The next funding period will focus on how direct BH3-only protein interaction explicitly regulates the BAX gateway to cell death. An emphasis will be placed on chemical and structural biology approaches to uncover the molecular details of the interaction surfaces between BAX and activating BH3-only proteins, and dissect the functional effects of these interactions in vitro and in cells. To achieve these goals, we will synthesize and deploy a panel of "stabilized alpha helices of BCL-2 domains" or SAHBs, which are structurally-stable and cell-permeable BH3 ligands that selectively target apoptotic proteins and reactivate apoptosis in vivo. Thus, we propose to use our novel chemical biology tools to (1) identify and characterize the interactions of BH3-only proteins BIM, BID, and PUMA with the mitochondrial executioner protein BAX, (2) determine the structural basis for the selective binding interactions between these BH3 death domains and BAX, and (3) investigate the physiologic role of BH3- mediated direct BAX activation in cells. We believe that by operating at the interface of chemistry, structural biology, apoptosis research, and cancer medicine, we are well-positioned to make major inroads into our understanding of the BAX-activation pathway and how it can be pharmacologically reactivated for the betterment of cancer patients. PUBLIC HEALTH RELEVANCE: At the very core of the cell's circuitry that dictates life and death lay an executioner protein named BAX, which is uniquely poised to inflict irreversible damage on the cell's power plant, the mitochondrion. Unlocking the mysteries of BAX regulation will impact the development of therapeutics to thwart unwanted cell death, such as in stroke, heart attack, and diabetes, and reactivate cell death in conditions marked by over-exuberant cellular survival, such as in autoimmune disease and cancer. We propose to use our innovative peptide-based compounds to probe the critical question of exactly how protein interaction triggers BAX to induce cell death. Ultimately, we aim to harness the fresh insights from these studies to generate prototype therapeutics that reactivate BAX-mediated apoptosis in human cancer.